Synthesis and Evaluation of Fluorine-18-Labeled L-Rhamnose Derivatives

The use of radiolabeled glucose for PET imaging resulted in the most commonly used tracer in the clinic, 2-deoxy-2-[18F]fluoroglucose (FDG). More recently, other radiolabeled sugars have been reported for various applications, including imaging tumors and infections. Therefore, in this study, we developed a series of fluorine-18-labeled L-rhamnose derivatives as potential PET tracers of various fungal and bacterial strains. Acetyl-protected triflate precursors of rhamnose were prepared and radiolabeled with fluorine-18 followed by hydrolysis to produce L-deoxy [18F]fluororhamnose. The overall radiochemical yield was 7–27% in a 90 min synthesis time with a radiochemical purity of 95%. In vivo biodistribution of the ligands using PET imaging showed that 2-deoxy-2-[18F]fluoro-L-rhamnose is stable for at least up to 60 min in mice and eliminated via renal clearance. The tracer also exhibited minimal tissue or skeletal uptake in healthy mice resulting in a low background signal.


Introduction
Structural imaging along with microbiological confirmation have long been the mainstay of the management of infectious diseases. However, structural imaging is nonspecific and in cases with deep seated infections, invasive methods such as biopsies or bronchoalveolar lavage are needed to provide microbiological analysis, sometimes with inconclusive results [1,2]. Positron emission tomography (PET) is a non-invasive imaging technique that measures the body's biochemical functions and metabolism. When coupled with CT or MRI, PET has been widely acknowledged in detecting tumors, infections, and systemic inflammatory diseases in different stages [3][4][5][6]. Fluorine-18 radiolabeled glucose, 2-deoxy-2-[ 18 F]fluoroglucose (FDG), is the most commonly used ligand in the clinic, mainly in cancer imaging. It has recently been approved for the imaging of infection and inflammation [7]. FDG is, however, unable to differentiate infectious etiologies from other causes of increased glucose metabolism, such as tumors and sterile inflammation.
More recently, other radiolabeled sugars have been developed for different purposes, such as imaging tumors [8] but more commonly for imaging different infections [9][10][11]. L-Rhamnose is a naturally occurring 6-deoxy sugar that is commercially used in the food, beverage, cosmetic and pharmaceutical industries ( Figure 1, compound 1) [12,13], but has no role in mammalian metabolism [14]. In E. coli, it is catabolized by a pathway involved with isomerization and phosphorylation [15], and in multiple fungal species, it is degraded through non-phosphorylated, aldolase glycolysis [16][17][18][19][20]. In addition, exclusively bacterial D-and L-sugars, including L-Rhamnose, are known to be incorporated into higher-order bacterial glycans [21].

Chemistry
Compound 3 reported by Liu et al. provided a base for this PET probe developmental work [22]. The fluorine on the 2-position of Compound 3 provided a suitable labeling site for fluorine-18, the most commonly used positron-emitting isotope for PET imaging studies. Inspired by the tracer-development work of FDG derivatives [23][24][25], the 3 and 6fluoro analogues of rhamnose were designed to compare their in vivo properties. The radiosynthesis of 5 was accomplished via nucleophilic substitution from the trifluoromethanesulfonate (triflate) precursor 14 followed by hydrolysis, similar to the synthesis of FDG [26]. The synthesis of the triflate precursor was achieved following a procedure found in the literature, with minor modifications (Scheme 1) [27]. Briefly, commercially available L-rhamnose (1) was used as the starting material. Per-O-acetylation of 1 through the iodine-promoted acetylation [28] resulted in a near-quantitative formation of pentaacetate 8. A two-steps selective O-deacetylation on the 2-position was performed according to the literature procedure [27]. The key steps to produce ortho−ester 10 were achieved via 1-bromination of compound 8 to produce compound 9, followed by a reaction with EtOH and 2,4,6-collidine. The bromo derivative 9 is unstable; therefore, the reaction to prepare 10 was performed immediately with the freshly prepared 9. The subsequent hydrolysis in HCl/acetone yielded the 2-hydroxyl derivative 11. To achieve the axial 2-fluorine in the desired product 5 through a nucleophilic substitution (SN2) reaction, an equatorial triflate is necessary for the precursor 14. Therefore, a stereo inversion of the 2-hydroxyl group of compound 11 was performed via a two-steps Lattrell-Dax epimerization reaction [29]. Compound 11 was converted to triflate derivative 12 first, followed by a nitrite-mediated  [22]. Their work has provided a platform for the development of radiolabeled L-rhamnose derivatives which might have potential for distinguishing infections from other pathologies and possibly distinguishing different pathogens (e.g., fungi vs. bacteria). Herein, we report our results in developing a series of L-rhamnose-based PET imaging probes ( Figure 1, compounds 5-7).

Chemistry
Compound 3 reported by Liu et al. provided a base for this PET probe developmental work [22]. The fluorine on the 2-position of Compound 3 provided a suitable labeling site for fluorine-18, the most commonly used positron-emitting isotope for PET imaging studies. Inspired by the tracer-development work of FDG derivatives [23][24][25], the 3 and 6-fluoro analogues of rhamnose were designed to compare their in vivo properties. The radiosynthesis of 5 was accomplished via nucleophilic substitution from the trifluoromethanesulfonate (triflate) precursor 14 followed by hydrolysis, similar to the synthesis of FDG [26]. The synthesis of the triflate precursor was achieved following a procedure found in the literature, with minor modifications (Scheme 1) [27]. Briefly, commercially available L-rhamnose (1) was used as the starting material. Per-O-acetylation of 1 through the iodine-promoted acetylation [28] resulted in a near-quantitative formation of pentaacetate 8. A two-steps selective O-deacetylation on the 2-position was performed according to the literature procedure [27]. The key steps to produce ortho−ester 10 were achieved via 1-bromination of compound 8 to produce compound 9, followed by a reaction with EtOH and 2,4,6-collidine. The bromo derivative 9 is unstable; therefore, the reaction to prepare 10 was performed immediately with the freshly prepared 9. The subsequent hydrolysis in HCl/acetone yielded the 2-hydroxyl derivative 11. To achieve the axial 2-fluorine in the desired product 5 through a nucleophilic substitution (S N 2) reaction, an equatorial triflate is necessary for the precursor 14. Therefore, a stereo inversion of the 2-hydroxyl group of compound 11 was performed via a two-steps Lattrell-Dax epimerization reaction [29]. Compound 11 was converted to triflate derivative 12 first, followed by a nitrite-mediated epimerization. The equatorial 2-hydroxyl epimer 13 was obtained in a reasonably good yield (65%). The desired triflate precursor 14 was synthesized quantitatively from compound 13. Synthesis of the reference non-radioactive standard, 2-deoxy-2-fluoro-L-Rhamnose (3) was initially attempted with the typical DAST fluorination of 13; however, no desired product was observed, probably due to the low stability of 13 under the reaction condition. With the milder TBAF fluorination condition mimicking the radiofluorination, 3 was obtained from its triflate precursor 14 in a 6% yield.  The synthesis of non−radioactive standard, 3-deoxy-3-fluoro-L-Rhamnose (23), and the radiolabeling precursor 24 were started from commercially available methyl-L-rhamnopyranoside 15 (Scheme 2). First, the 3-hydroxyl group was selectively protected to achieve benzyl derivative 16 using a dimethyltin dichloride catalyzed reaction. Acetylation on the rest of the hydroxyl groups was performed to obtain 17. Without purification, the 1-methoxy group on crude compound 17 was converted to acetate to produce 18 in a good yield over two-steps. Subsequent de-benzylation with palladium on carbon produced 3-hydroxyl compound 19. A similar Lattrell-Dax epimerization as described above for the synthesis of compound 13 was performed to obtain the stereo-inverted epimer 21. Fluorination with DAST, followed by de-acetylation, successfully produced 23 in a moderate yield. The triflate precursor 24 was synthesized by treating 21 with triflic anhydride/pyridine using a standard condition. The synthesis of non−radioactive standard, 3-deoxy-3-fluoro-L-Rhamnose (23), and the radiolabeling precursor 24 were started from commercially available methyl-L-rhamnopyranoside 15 (Scheme 2). First, the 3-hydroxyl group was selectively protected to achieve benzyl derivative 16 using a dimethyltin dichloride catalyzed reaction. Acetylation on the rest of the hydroxyl groups was performed to obtain 17. Without purification, the 1-methoxy group on crude compound 17 was converted to acetate to produce 18 in a good yield over two-steps. Subsequent de-benzylation with palladium on carbon produced 3-hydroxyl compound 19. A similar Lattrell-Dax epimerization as described above for the synthesis of compound 13 was performed to obtain the stereo-inverted epimer 21. Fluorination with DAST, followed by de-acetylation, successfully produced 23 in a moderate yield. The triflate precursor 24 was synthesized by treating 21 with triflic anhydride/pyridine using a standard condition.
Synthesis of non−radioactive standard, 6-fluoro-L-Rhamnose (29), and the triflate precursor 30 (Scheme 3) was more straight-forward. Starting from commercially available L-mannose 25, selective protection of the primary hydroxyl group on 6-CH 2 OH was achieved using triphenylmethyl chloride, followed by a per-O-acetylation to provide 26. The triphenylmethyl protective group was then removed to obtain 27. The non-radioactive standard 29, was obtained from the reaction of 27 with DAST, followed by de-acetylation under the same condition as above. Similarly, the triflate precursor 30 was synthesized by treating the hydroxyl derivative 27 with triflic anhydride/pyridine. Synthesis of non−radioactive standard, 6-fluoro-L-Rhamnose (29), and the triflate precursor 30 (Scheme 3) was more straight-forward. Starting from commercially available L-mannose 25, selective protection of the primary hydroxyl group on 6-CH2OH was achieved using triphenylmethyl chloride, followed by a per-O-acetylation to provide 26. The triphenylmethyl protective group was then removed to obtain 27. The non-radioactive standard 29, was obtained from the reaction of 27 with DAST, followed by de-acetylation under the same condition as above. Similarly, the triflate precursor 30 was synthesized by treating the hydroxyl derivative 27 with triflic anhydride/pyridine.   Synthesis of non−radioactive standard, 6-fluoro-L-Rhamnose (29), and the triflate precursor 30 (Scheme 3) was more straight-forward. Starting from commercially available L-mannose 25, selective protection of the primary hydroxyl group on 6-CH2OH was achieved using triphenylmethyl chloride, followed by a per-O-acetylation to provide 26. The triphenylmethyl protective group was then removed to obtain 27. The non-radioactive standard 29, was obtained from the reaction of 27 with DAST, followed by de-acetylation under the same condition as above. Similarly, the triflate precursor 30 was synthesized by treating the hydroxyl derivative 27 with triflic anhydride/pyridine.

Radiochemistry
The radiosyntheses of the target probes 5-7 were achieved in two-steps: fluorination and the deprotection of the acetyl-protecting group (Scheme 4). Labeling efficiency was first standardized manually using 3-10 mCi of [ 18 F]fluoride. In general, the cyclotron-produced [ 18 F]fluoride was trapped on an anion exchange cartridge, and was eluted with a water/methanolic solution of tetrabutylammonium bicarbonate (TBAB) [30].
TBAB was selected because it was reported to be more efficient for the radiofluorination of base-sensitive precursors. After azeotropic drying with anhydrous acetonitrile, the precursor solution was added and the radiofluorination was performed at 70 • C. The progress of the reaction was monitored using analytical high-performance liquid chromatography (HPLC). The fluorine-18-labeled intermediate was purified via HPLC using a semi-preparative column.

Radiochemistry
The radiosyntheses of the target probes 5-7 were achieved in two-steps: fluorination and the deprotection of the acetyl-protecting group (Scheme 4). Labeling efficiency was first standardized manually using 3-10 mCi of [ 18 F]fluoride. In general, the cyclotron-produced [ 18 F]fluoride was trapped on an anion exchange cartridge, and was eluted with a water/methanolic solution of tetrabutylammonium bicarbonate (TBAB) [30]. TBAB was selected because it was reported to be more efficient for the radiofluorination of base-sensitive precursors. After azeotropic drying with anhydrous acetonitrile, the precursor solution was added and the radiofluorination was performed at 70 °C. The progress of the reaction was monitored using analytical high-performance liquid chromatography (HPLC). The fluorine-18-labeled intermediate was purified via HPLC using a semi-preparative column. The collected fraction was diluted with water and trapped on a solid phase extraction (SPE) cartridge. The trapped intermediate was eluted with ethanol through a potassium carbonate cartridge into vial containing a stir bar. The eluted mixture was stirred for 10 min and neutralized. The final product was retained on an FDG purification cartridge and eluted with normal saline. The radiochemical yields (RCYs) for 5-7 were 7-12%, 10-15%, and 18-25%, respectively (uncorrected, n > 3), in ~90 min. Radiochemical purities were >95%. The identity of the final radiolabeled product was confirmed via liquid chromatography mass spectrometry (LCMS) by co-eluting with a non-radioactive standard. A representative HPLC for compound 6 is presented in Figure 2. A radio TLC analysis revealed the absence of [ 18   The collected fraction was diluted with water and trapped on a solid phase extraction (SPE) cartridge. The trapped intermediate was eluted with ethanol through a potassium carbonate cartridge into vial containing a stir bar. The eluted mixture was stirred for 10 min and neutralized. The final product was retained on an FDG purification cartridge and eluted with normal saline. The radiochemical yields (RCYs) for 5-7 were 7-12%, 10-15%, and 18-25%, respectively (uncorrected, n > 3), in~90 min. Radiochemical purities were >95%. The identity of the final radiolabeled product was confirmed via liquid chromatography mass spectrometry (LCMS) by co-eluting with a non-radioactive standard. A representative HPLC for compound 6 is presented in Figure 2. A radio TLC analysis revealed the absence of [ 18 F]fluoride in the labeled intermediate and the final product ( Figure 3).
After successful manual standardization of the procedure, an automated synthesis was performed in a GE Tracerlab FX-N module ( Figure 4). The synthesis consisted of seven reagent vials. Vials 1-5 were used for the elution of [ 18 F]fluoride, drying, and fluorination reaction. Vials 13 and 14 were used for the trap and release of the purified intermediate. The valve 15 normal close was connected to the crude product vial through an inline K 2 CO 3 cartridge for hydrolysis. The crude product was formulated by trapping on an FDG cartridge and eluting with normal saline.
All three tracers were stable in saline (pH 6) at room temperature for at least 4 h. Compound 5 was stable at 37 • C in whole human serum and compound 7 was slowly decomposed (Table 1). However, compound 6 rapidly decomposed resulting in 70% being intact after 4 h ( Figure S1). Aqueous phase: 95% water + 5% ACN with 0.1% NH4OH; organic phase: 95% ACN + 5% water with 0.1% NH4OH.  All three tracers were stable in saline (pH 6) at room temperature for at least 4 h. Compound 5 was stable at 37 °C in whole human serum and compound 7 was slowly decomposed (Table 1). However, compound 6 rapidly decomposed resulting in 70% being intact after 4 h ( Figure S1).

PET/CT Imaging
Representative dynamic PET images for a healthy mouse injected with a single bolus of 2-deoxy-2-[ 18 F]fluoro-L-rhamnose (5) and the averaged time activity curves are shown in Figure 5A,B. The images indicate that after the initial blood pool clearance, the tracer shows little to no tissue uptake in the lungs, heart, liver, spleen, or brain and is quickly eliminated by renal clearance. These results suggest that 5 is not metabolized by mammalian cells, as expected. Additionally, it was not detected within the skeletal system at any point and thus the ligand is stable for at least 60 min in vivo. The time activity curves showed that 5 has a short biological half-life of 3.4 ± 1.7 min. The in vivo biodistribution

PET/CT Imaging
Representative dynamic PET images for a healthy mouse injected with a single bolus of 2-deoxy-2-[ 18 F]fluoro-L-rhamnose (5) and the averaged time activity curves are shown in Figure 5A,B. The images indicate that after the initial blood pool clearance, the tracer shows little to no tissue uptake in the lungs, heart, liver, spleen, or brain and is quickly eliminated by renal clearance. These results suggest that 5 is not metabolized by mammalian cells, as expected. Additionally, it was not detected within the skeletal system at any point and thus the ligand is stable for at least 60 min in vivo. The time activity curves showed that 5 has a short biological half-life of 3.4 ± 1.7 min. The in vivo biodistribution of the other two radiolabeled derivatives of rhamnose (6 and 7) were also obtained using dynamic PET imaging. Both 6 and 7 showed rapid defluorination upon injection in mice, as evidenced by the increasing skeletal uptake in the representative PET images (Figure 6). of the other two radiolabeled derivatives of rhamnose (6 and 7) were also obtained using dynamic PET imaging. Both 6 and 7 showed rapid defluorination upon injection in mice, as evidenced by the increasing skeletal uptake in the representative PET images ( Figure  6).

General
Methyl-rhamnopyranoside was obtained from Combi-Blocks Inc.

General
Methyl-rhamnopyranoside was obtained from Combi-Blocks Inc. (San Diego, CA, USA). All other chemicals and solvents were received from Sigma Aldrich (St. Louis, MO, USA) and used without further purification. Fluorine-18 was received from the National Institutes of Health cyclotron facility (Bethesda, MD, USA). A Chromafix PS-HCO3 cartridge was purchased from Synthra GmbH (Hamburg Germany). Oasis HLB plus and K 2 CO 3 cartridges were obtained from Waters (Milford, MA, USA). An FDG cartridge was purchased from NucMedCor (San Francisco, CA, USA). Mass spectrometry (MS) was performed on a 6130 Quadrupole LC/MS, Agilent Technologies instrument equipped with a diode array detector. 1 H, 13 C, and 19 F NMR spectra were recorded on a Varian spectrometer (400 MHz). Chemical shifts (ppm) were reported relative to the solvent residual peaks. HPLC for purification and analytical analysis were performed on an Agilent 1200 Series instrument equipped with multi-wavelength detectors along with a flow count radiodetector (Eckert & Ziegler, B-FC-3500 diode). LC-MS/MS analysis was performed on an Agilent 6460C triple quadrupole mass spectrometer with an ESI source and a flow count radiodetector (Eckert & Ziegler, B-FC-3500 diode). The LC unit was an Agilent 1200 series chromatographic system equipped with a 1260 binary pump, 1290 thermostatted column compartment and 1260 high-performance autosampler. Instrument control and data processing were performed using Aglient's MassHunter Software.

Radiochemical Syntheses
All three tracers were prepared following the general procedure described below. Radiosyntheses were performed on a GE Tracerlab FX-N2 synthesizer. The synthesis consisted of 7 reagent vials on the GE synthesizer. Vials 1-5 were used for the elution, the drying of fluorine-18, and the fluorination reaction. Vials 13-14 were used for the formulation of the purified intermediate. An inline K 2 CO 3 cartridge was incorporated between valve 15 and the product vial. Specifically, the reagent vials contained the following: Vial 1: tetrabutylammonium bicarbonate solution (150 µL, 0.075 M), 50 µL water, and MeOH (1 mL); Vial 2: ACN (1 mL); Vial 3: triflate precursor (5 mg) in ACN (0.6 mL); Vial 4: water (1 mL); Vial 5: HPLC solvent (2.0 mL); Vial 13: EtOH (2 mL); Vial 14: water (6 mL); HPLC dilution flask: water (30 mL). The vial 11 inlet port was connected to the V15 right port to transfer the intermediate to reactor 2 (R2). Typically, 7.4 GBq (200 mCi) [ 18 F]fluoride in 2.5 mL of water was passed through a Chromafix PS-HCO 3 cartridge, which was rinsed with 1 mL of acetonitrile. The retained [ 18 F]fluoride was eluted from the cartridge into reactor 1 (R1) with the eluent in Vial 1 and dried under a N 2 /vacuum at 75 • C for 4 min. R1 was cooled to 50 • C, acetonitrile in Vial 2 was added and the activity was azeotropically dried at 55 • C for 3 min and at 95 • C for an additional 3 min under a N 2 /vacuum. The activity was further dried using a vacuum for 3 min. The [ 18 F]fluoride drying cycle took about 20 min.
The triflate precursor solution in Vial 3 was added to the dried activity. The resulting solution was stirred at 70 • C for 20 min and cooled to 45 • C. The reaction mixture was diluted with 1.0 mL of water (Vial 4) and transferred into Tube 2. R1 was rinsed with HPLC mobile phase (Vial 5) and the solution was also transferred into Tube 2. The solution in Tube 2 was thoroughly mixed by bubbling N 2 for 10 s and injected into the HPLC for purification. HPLC condition: Phenomenex Luna (2) C18 column, 250 × 10 mm, 5 µm. Mobile phase: 40% ACN in 0.1% TFA. Flow rate: 4 mL/min. The labeled intermediate was eluted for about 12-14 min which was collected in the dilution flask containing 30 mL of water and passed through an Oasis HLB plus cartridge (pre-conditioned with 5 mL of ethanol, 10 mL of air, and 10 mL of water). The trapped labeled intermediate was rinsed with 6 mL of water (Vial 14), eluted with 2 mL of absolute ethanol (Vial 13) through the inline K 2 CO 3 cartridge to the vial with a stir bar, and then the mixture was stirred for 10 min. Saline pH 2.5 (2 mL) was added to the vial, the solution was passed through a preconditioned (18 mL ethanol and 30 mL water) FDG cartridge. The cartridge was washed with saline pH 2.5 (1 mL). The product was eluted with 4 mL of normal saline. The synthesis time was~90 min.

Animals
All experimental procedures, including the handling and care of the animals, were approved by the Animal Care and Use Committee of the Clinical Center of the NIH and performed under relevant NIH policies. Healthy female CD-1 mice (aged 6-8 weeks, Charles River, Charleston, SC, USA) were used for the PET/CT imaging. All mice were housed with a 12-h light/dark cycle with free access to food and water.

PET/CT Imaging
The mice were first anesthetized with 3-4% isoflurane and the animals were kept warm using a heating pad during the scan.
A preclinical Inveon PET/CT scanner (Siemens Medical Solutions, Malvern, PA, USA) was used. Each animal was first secured onto the scanner bed and placed symmetrically within the center FOV. A CT scan was then performed for attenuation correction and anatomic guidance when placing the volumes of interest for the calculation of radioactivity concentrations (VOIs). Immediately after the PET acquisition was started, the tracer was injected via the tail vein (~9 MBq) as a bolus (n = 4), followed by a quick saline flush (total~200 µL). Dynamic PET images were acquired over a period of 60 min in list mode. The emission sinograms were corrected for scatter, 18 F-decay, random, and dead time. The resulting histograms were then reconstructed by applying Fourier rebinning and the 3D ordered subject expectation maximization algorithm (OSEM-3D). The images were analyzed with pMOD 3.2. For dynamic PET imaging with 3-deoxy-3-[ 18 F]-fluoro-Lrhamnose, a Mediso PET/CT scanner was used. The imaging methodology and the dose administered did not differ from the previous scans conducted on the Inveon imager. The images were analyzed using Fusion software (Mediso Ltd., Budapest, Hungary).

Conclusions
Three fluorine-18-labeled L-rhamnose derivatives have been developed for PET imaging studies with the ultimate goal of using those compounds to image various infectious diseases. Using triflate precursors, these new radioligands were prepared with medium to high RCY. The radiolabeling methods were successfully automated with the intention of large-scale production. All three derivatives were used for PET/CT imaging in mice. Of these, 3-deoxy-3-[ 18 F]fluoro-L-rhamnose and 6-[ 18 F]fluoro-L-rhamnose showed rapid defluorination after in vivo injection ( Figure 6). On the other hand, 2-deoxy-2-[ 18 F]fluoro-Lrhamnose was found to be very stable upon injection in healthy mice. This ligand rapidly cleared and there was minimal uptake in various organs indicating lack of uptake and/or metabolism by mammalian cells. There was also no liver metabolism detected and excretion was completely via the renal route ( Figure 5). Furthermore, unlike FDG, there was no discernable accumulation of the ligand in the myocardial tissues. We are only describing uptake of radiolabeled L-Rhamnose in healthy animals. We are currently finalizing in vitro and in vivo uptake results in different animal models of infection in order to establish the usefulness of radiolabeled Rhamnose in detecting infection in vivo. The results will be published separately.